Investigation of multiple-dynein transport of melanosomes by non-invasive force measurement using fluctuation unit χ

Abstract

Pigment organelles known as melanosomes disperse or aggregate in a melanophore in response to hormones. These movements are mediated by the microtubule motors kinesin-2 and cytoplasmic dynein. However, the force generation mechanism of dynein, unlike that of kinesin, is not well understood. In this study, to address this issue, we investigated the dynein-mediated aggregation of melanosomes in zebrafish melanophores. We applied the fluctuation theorem of non-equilibrium statistical mechanics to estimate forces acting on melanosomes during transport by dynein, given that the energy of a system is related to its fluctuation. Our results demonstrate that multiple force-producing units cooperatively transport a single melanosome. Since the force is generated by dynein, this suggests that multiple dyneins carry a single melanosome. Cooperative transport has been reported for other organelles; thus, multiple-motor transport may be a universal mechanism for moving organelles within the cell.

Effects of the dynein inhibitor dynarrestin on the number of motor proteins transporting synaptic cargos

“…Note that direction in motion was set to the positive direction when χ was calculated. The discreteness of χ was found with synaptic cargo transport and was observed previously for other cargo transports(Hasegawa et al, 2019; Hayashi, Hasegawa, et al, 2018; Hayashi, Tsuchizawa, et al, 2018). After applying a classification method (Methods), we found 6 clusters for both anterograde (Figure 3A) and retrograde (Figure 4A) transport.…”

“…The peak of P (Δ X ) shifts to the right because of the directional motion of transported cargo, and variance increases due to the diffusional effect, as Δ t becomes larger. P (Δ X ) was well described by a Gaussian function as well as those measured for other intracellular cargos (Hasegawa et al, 2019; Hayashi, Hasegawa, et al, 2018; Hayashi, Tsuchizawa, et al, 2018). Note that the abnormal diffusion property (i.e., P (Δ X ) is not a Gaussian function) often reported for intercellular cargo transport(Fakhri et al, 2014; Posey, Blaisdell-Pijuan, Knoll, Saif, & Ahmed, 2018; Shin et al, 2019; Tabei et al, 2013) appeared upon analysis of the whole trajectory without dividing it into CVSs(Hayashi et al, 2013).…”

“…Based on the fluctuation theorem(Ciliberto, Joubaud, & Petrosyan, 2010; Evans, Cohen, & Morriss, 1993), the relationship between χ and the driving force ( F ) is represented as where k B is the Boltzmann constant and T eff is an effective temperature(Hasegawa et al, 2019; Hayashi, Tsuchizawa, et al, 2018), which is a generalized temperature in a non-equilibrium system(Crisanti & Ritort, 2003; Cugliandolo, 2011; Hayashi & Sasa, 2004). In our previous studies on intracellular cargo transport(Hasegawa et al, 2019; Hayashi, Hasegawa, et al, 2018; Hayashi, Tsuchizawa, et al, 2018), T eff was not equal to the temperature of the environment ( T ), due to non-equilibrium effects. When one anterograde FPU corresponded to a kinesin dimer (which has a stall force of about 5 pN), T eff was estimated to be about 10 times k B T for synaptic cargo transport.…”

“…The in vivo driving force produced by motor proteins acting on intracellular cargo was investigated recently with non-invasive force measurements based on non-equilibrium statistical mechanics(Hasegawa, Sagawa, Ikeda, Okada, & Hayashi, 2019; Hayashi, 2018; Hayashi, Hasegawa, Sagawa, Tasaki, & Niwa, 2018; Hayashi, Matsumoto, Miyamoto, & Niwa, 2019; Hayashi, Tsuchizawa, Iwaki, & Okada, 2018). With the reported method, inferring the driving force from the fluctuating motion of transported cargo, the number of force producing units (FPUs), compromising motor proteins carrying a single cargo, was estimated to be 1-3 anterograde FPUs for synaptic vesicle precursor transport in motor neurons of Caenorhabditis elegans (Hayashi, Hasegawa, et al, 2018), 1-4 anterograde and 1-3 retrograde FPUs for endosome transport in mice dorsal ganglion neurons(Hayashi, Tsuchizawa, et al, 2018), and 1-5 retrograde FPUs for melanosome transport in zebrafish melanophores.…”

Physical parameters describing neuronal cargo transport by kinesin UNC-104

“…6b), when the viscosity effect is high enough. This suggests that long time courses should be divided into several constant velocity segments (CVSs) when the number of motors transporting a cargo is an object to be measured (Hasegawa et al 2019;Hayashi 2018;Hayashi et al 2018a;Hayashi et al 2018b).…”